The four nucleotide bases of DNA molecules carry genetic information. This information, in the form of codons of three contiguous bases is transcribed by mRNA and translated by tRNA and ribosomes to form proteins. The genetic code is the relation between a triplet codon and a particular amino acid. Of the sixty-four possible codon triplets which form the genetic code, there are three stop or terminating codons which are known to stop protein production at cellular ribosomes; the other sixty-one triplets in the code correspond to one or another amino acid. See Table 1
TABLE 1UUUPheUCUSerUAUTyrUGUCysUUCPheUCCSerUACTyrUGCCysUUALeuUCASerUAAStopUGAStopUUGLeuUCGSerUAGStopUGGTrpCUULeuCCUProCAUHisCGUArgCUCLeuCCCProCACHisCGCArgCAULeuCCAProCAAGlnCGAArgCUGLeuCCGProCAGGlnCGGArgAUULleACUThrAAUAsnAGUSerAUCLleACCThrAACAsnAGCSerAUALleACAThrAAALysAGAArgAUGMetACGThrAAGLysAGGArgGUUValGCUAlaGAUAspGGUGlyGUCValGCCAlaGACAspGGCGlyGUAValGCAAlaGAAGluGGAGlyGUGValGCGAlaGAGGluGGGGly
When genetic instructions are translated at ribosomes, the amino acids are strung together to form complex polypeptides. However, when a stop codon is read, it is interpreted as a stop signal terminating the protein production. The three stop codons are UAG (amber), UAA (ochre) and UGA (opal). Mutations that change a codon to stop codon are called nonsense mutations and, as a result, genetic phenotypes may not be expressed. Thus, despite the presence of a gene directing expression, a crucial protein may not be produced because an unwanted stop signal reaches a ribosome and terminates an unfinished protein.
Transfer RNAs (tRNAs) translate mRNA into a protein on the ribosome. Each transfer RNA contains an anti-codon region that hybridizes with mRNA, and an amino acid which may be attached to the growing peptide. The structural gene of tRNA is about 72–90 nucleotides long and folds into a cloverleaf structure. tRNAs are transcribed by RNA polymerase III and contain their own intragenic split promoters that become a part of the mature tRNA coding sequence (Sharp S. J., Schaack J., Coolen L., Burke D. J. and Soll D., “Structure and transcription of eukaryotic tRNA genes”, Crit. Rev. Biochem, 19:107–144 (1985); Geiduschek E. O., and Tocchini-Valentini, “Transcription by RNA polymerase III”, Annu. Rev. Biochem. 57:873–914 (1988)).
Nonsense suppressors are alleles of tRNA genes that are altered in the anticodon so that they can insert an amino acid in response to a termination codon. For example, an ochre mutation results in the creation of a UAA codon in messenger RNA. An ochre suppressor gene produces tRNA with a AUU anticodon that inserts an amino acid at the UAA site permitting continued translation despite the presence of a nonsense codon.
A number of nonsense suppressor tRNA alleles have been identified in prokaryotes and eukaryotes such as yeast and C. elegans. However to date, no mammalian cell line containing functional suppressor tRNA has been isolated using classical genetic selection. Attempts to isolate suppressor tRNAs from higher eukaryotes resulted in the identification of an opal suppressor phosphoserine tRNA in the chicken genome (Hatfield D. L., Dudock B. S., and Eden F. C., “Characterization and nucleotide sequence of a chicken gene encoding an opal suppressor tRNA and its flanking DNA segments”, Proc. Natl. Acad. Sci. U.S.A., 80:4940–4944 (1983)), and later in the human genome (O'Neill V. A., Eden F. C., Pratt K., and Hatfield D. L., “A human opal suppressor tRNA gene and pseudogene”, J. Biol. Chem. 260:2501–2508 (1985)). The two differ from each other at only a single nucleotide position. Suppressor tRNAs may also cause readthrough of the naturally occurring stop codons, thereby producing extended proteins with altered functions. Suppression of termination may be deleterious to the cell, although multiple natural stop codons at the end of the gene may provide safeguard from such harmful effects. The different suppressor tRNAs vary in their suppression efficiency. In E. coli and other systems the amber suppressors are relatively more efficient, ochre suppressors are less efficient while opal are the least, this suggests that the amber codons are used infrequently to terminate protein synthesis, while ochre and opal codons are more frequently used as natural termination signals.
Restoration of a normal phenotype by suppressors will depend on the type of amino acid inserted at the position of the nonsense codon. The inserted amino acid may be incompatible with the structure, function or stability of the gene product. Hence, there exists a need for a wide variety of suppressor tRNAs to insert different amino acids. Amber and ochre suppressors derived from a Xenopus Laevis tyrosine tRNA gene were shown to be functional in mammalian cells in transient transfection assays as well as in permanent cell lines (Laski F. A., Belagaje U. L., RajBhandary U. L. and Sharp P. A., “An amber suppressor tRNA gene derived by site-directed mutagenesis: cloning and expression in mammalian cells”, Proc. Natl. Acad. Sci. USA, 79:5813–5817 (1982); Laski F. A., Belagaje R., Hudzoal R. M., Capecchi M. R., Palese P., RajBhandary U. L. and Sharp P. A., “Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells”, EMBO J 3:2445–2452 (1984); Hudziak R. M., Laski R. A., RajBhandary U., Sharp, P. A. and Capecchi M. R., “Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor transfer RNA genes”, Cell 31:131–146 (1982)). Capone and co-workers similarly generated amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene Capone J. P., Sharp P. A. and RajBhandary U. L., “Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene”, EMBO J 4:213–221 (1985)).
In addition to permitting read-through of a mutation which causes a nonsense codon in the middle of a transcribed protein sequence, there are also times when one wants to manipulate a translation to truncate gene products. In either case, there exists a need for a suppression mechanism which would permit the cellular ribosomes to ‘read through’ such stop signals when they are unwanted. There is also a need for the opportunity to site specifically modify protein synthesis by deliberately altering the translation of the genetic code to learn about protein function.
It is an object of the present invention to provide novel nonsense suppressor tRNA's which are functional in cells and methods of use of the same in genetic engineering protocols.